Gene expression, defined as the conversion of the nucleotide sequence of a gene into the nucleotide sequence of a stable RNA or into the amino acid sequence of a protein, is very tightly regulated in every living organism. Regulation of gene expression both of mRNA stability and translation is important in cellular responses to development or environmental stimuli such as nutrient levels, cytokines, hormones, and temperature shifts, as well as environmental stresses like hypoxia, hypocalcemia, viral infection, and tissue injury (reviewed in Guhaniyogi & Brewer, 2001, Gene 265(1-2):11-23). Furthermore, alterations in mRNA stability have been causally connected to specific disorders, such as neoplasia, thalassemia, and Alzheimer's disease (reviewed in Guhaniyogi & Brewer, 2001, Gene 265(1-2):11-23 and Translational Control of Gene Expression, Sonenberg, Hershey, and Mathews, eds., 2000, CSHL Press).
Giordano et al., U.S. Pat. No. 6,558,007 (hereafter referred to as “the '007 patent”), assert that they provide a screening assay using a 5′ mRNA UTR biased cDNA library or a 3′ mRNA UTR biased cDNA library. The '007 patent further asserts that they provide a method of identifying a regulatory UTR sequence using their 5′ or 3′ mRNA UTR biased cDNA libraries. The '007 patent does not provide assays that mimic the in vivo state of a gene controlled by the presence of more than one UTR, for example, genes which are flanked by a 5′ UTR and a 3′ UTR. Moreover, the approach of the '007 patent requires the libraries described therein.
Pesole et al. assert that the 5′- and 3′-UTRs of eukaryotic mRNAs are known to play a crucial role in post-transcriptional regulation of gene expression. Pesole et al., (2002) Nucleic Acids Research, 3(1):335-340, which is hereby incorporated by reference in its entirety. They develop and describe several databases with nucleic acid sequences from UTRs. Many of their database entries are enriched with specialized information including the presence of sequence patterns demonstrated by experimental evidence to play a functional role in gene regulation. Pesole et al. do not provide assays to obtain such experimental evidence, nor do they suggest that such experiments mimicked the in vivo state of the UTR database entry. Moreover, the methodology of Pesole et al. is based on sequence analysis and prior experimental evidence. Pesole et al. do not provide experimental screening methods for developing agents to modulate the 5′- and 3′-UTRs of eukaryotic mRNAs that are known to play a crucial role in post-transcriptional regulation of gene expression nor do they suggest a methodology to find novel 5′- and 3′-UTRs of eukaryotic mRNAs that play a crucial role in post-transcriptional regulation of gene expression. In addition, the approach of Pesole et al. requires the databases described therein.
Trotta et al. assert that a the interaction of the La antigen with mdm2 5′ UTR enhances mdm2 mRNA translation. Trotta et al., (2003) Cancer Cell 3:145-160, which is hereby incorporated by reference in its entirety. They do not suggest methods or agents to screen or identify more UTRs with a similar role in translational regulation of gene expression. Moreover, no agents are provided to screen for compounds that would modulate the regulation of mdm2 mRNA translation.